Desulfitobacterium strains have the ability to dechlorinate halogenated compounds under anaerobic conditions by dehalorespiration. The complete genome of the tetrachloroethene (PCE)-dechlorinating strain Desulfitobacterium hafniense Y51 is a 5,727,534-bp circular chromosome harboring 5,060 predicted protein coding sequences. This genome contains only two reductive dehalogenase genes, a lower number than reported in most other dehalorespiring strains. More than 50 members of the dimethyl sulfoxide reductase superfamily and 30 paralogs of the flavoprotein subunit of the fumarate reductase are encoded as well. A remarkable feature of the genome is the large number of O-demethylase paralogs, which allow utilization of lignin-derived phenyl methyl ethers as electron donors. The large genome reveals a more versatile microorganism that can utilize a larger set of specialized electron donors and acceptors than previously thought. This is in sharp contrast to the PCE-dechlorinating strain Dehalococcoides ethenogenes 195, which has a relatively small genome with a narrow metabolic repertoire. A genomic comparison of these two very different strains allowed us to narrow down the potential candidates implicated in the dechlorination process. Our results provide further impetus to the use of desulfitobacteria as tools for bioremediation.Halogenated organic compounds are released into the environment from natural and anthropogenic sources. Many anthropogenic halogenated chemicals, like chlorinated haloalkenes (7, 10, 46), benzenes (1), and dioxins (5), are of particular concern due to their toxicity to humans and other forms of life. This toxicity is often paired with high recalcitrance to degradation, especially in anaerobic environments, leading to persistent contamination.Anaerobic environments are frequently characterized by limited availability of electron acceptors. Theoretical calculations have shown that coupling the reduction of many halogenated organic compounds to the oxidation of suitable substrates is a way to harness energy (46). As determined two decades ago, this source of energy is utilized by the microbial community. The oxidation of available electron donors coupled to the reduction of halogenated organic compounds while energy is conserved is called dehalorespiration (7,10,46). Dehalorespiring strains have been isolated independently from contaminated sites around the world. The two most prominent genera resulting from these isolation efforts are Dehalococcoides (29) and Desulfitobacterium (51), and various strains of these genera are used as model systems to study dehalorespiration (8,11,51).Dehalococcoides ethenogenes 195 is one of the few strains isolated to date which can dechlorinate tetrachloroethene (PCE) to ethene (29). D. ethenogenes 195 can use only hydrogen as an electron donor and chlorinated compounds as electron acceptors (29).Desulfitobacterium strains are also known to dechlorinate a wide variety of substrates, including halophenolic compounds and chloroalkenes (7,10,46). Although several s...

Membrane-bound ATPase was found in membranes of the archaebacterium Methanosarcina barkeri. The ATPase activity required divalent cations, Mg2' or Mn2 , and maximum activity was obtained at pH 5.2. The activity was specifically stimulated by HS03-with a shift of optimal pH to 5.8, and N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. The enzyme could be solubilized from membranes by incubation in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. The solubilized ATPase was purified by DEAE-Sepharose and Sephacryl S-300 chromatography. The molecular weight of the purified enzyme was estimated to be 420,000 by gel filtration through Sephacryl S-300. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate revealed two classes of subunit, Mr 62,000 (a) and 49,000 (Ii) associated in the molar ratio 1:1. These results suggest that the ATPase of M. barkeri is similar to the FoF, type ATPase found in many eubacteria.Methanogenic bacteria are strictly anaerobic organisms, and most species are able to grow autotrophically with H2 and CO2 as substrates for methanogenesis. On the basis of studies of the 16S RNA sequence, they are considered to belong to the archaebacteria (1). Other properties of methanogens are also different from those of the eubacteria; the membranes of methanogens contain ether-linked polyisoprenoid glycerol lipids (30) and their cell walls lack peptidoglycan (14). Apart from methanogens, the archaebacteria include extreme halophiles and thermoacidophiles.In the chemiosmotic mechanism of Mitchell (20), a proton motive force established across the cell membrane is a driving force for ATP synthesis. An H+-translocating ATPase (FoF1 ATPase) which catalyzes ATP synthesis has been observed in membranes of both eucaryotes and eubacteria (26). Although membrane-bound ATPase activities were found in membranes of thermoacidophilic archaebacteria, Thermoplasma (28), and Sulfolobus (32) Solubilization and purification of the ATPase. The membrane fraction was suspended in 1 mM Tris-maleate buffer (pH 6.9) containing 0.5 mM EDTA. After incubation at 8°C for 1 h with gentle stirring, the suspension was centrifuged at 100,000 x g for 90 min. The supernatant (42 ml, 2.4 mg of protein per ml) was applied to a DEAE-Sepharose column (2.6 by 20 cm) equilibrated with 50 mM Tris-maleate (pH 6.9) (column buffer). After the column had been washed with one column volume of the column buffer, a linear gradient from 0 to 0.6 M NaCl in 50 mM Tris-maleate buffer (pH 6.9) was run at 8°C. The fractions containing ATPase activity were pooled (15 ml), and solid ammonium sulfate was added up to reach 65% saturation. The precipitate obtained after centrifugation at 10,000 x g for 20 min was dissolved in a minimal volume of 50 mM Tris-maleate buffer (pH 6.9), and the solution was centrifuged to remove any insoluble material. The supernatant was then applied to a Sephacryl S-300 column (1.6 by 80 cm) equilibrated with the column buffer. The flow rate was 10 ml/min at 8°C, and the fractions with ATPase activity were collec...

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Desulfitobacterium hafniense Y51 is a dechlorinating bacterium that encodes an unusually large set of O-demethylase paralogs and specialized respiratory systems including specialized electron donors and acceptors. To use this organism in bioremediation of tetrachloroethene (PCE) or trichloroethene (TCE) pollution, expression patterns of its 5,060 genes were determined under different conditions using 60-mer probes in DNA microarrays. PCE, TCE, fumarate, nitrate, and dimethyl sulfoxide (DMSO) respiration all sustain the growth of strain Y51. Global transcriptome analyses were thus performed using various electron donor and acceptor couples (respectively, pyruvate and either fumarate, TCE, nitrate, or DMSO, and vanillate/fumarate). When TCE is used as terminal electron acceptor, resulting in its detoxification, a series of electron carriers comprising a cytochrome bd-type quinol oxidase (DSY4055-4056), a ferredoxin (DSY1451), and four Fe-S proteins (DSY1626, DSY1629, DSY0733, DSY3309) are upregulated, suggesting that the products of these genes are involved in PCE oxidoreduction. Interestingly, the PCE dehalogenase cluster (pceABCT) is constitutively expressed in the media tested, with pceT being upregulated and pceC downregulated in pyruvate/TCE-containing medium. In addition, another dehalogenation enzyme (DSY1155 coding for a putative chlorophenol reductive dehalogenase), is induced 225-fold in that medium, despite not being involved in PCE respiration. Remarkably since the reducing equivalents formed during pyruvate conversion to acetyl-CoA are channeled to electron acceptors including halogenated compounds, pyruvate induces expression of a pyruvate:ferredoxin oxidoreductase. This study paves the way to understanding the physiology of D. hafniense, optimizing this microbe as a bioremediation agent, and designing bioarray sensors to monitor the presence of dechlorinating organisms in the environment.

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